Evaluación de formulaciones inmunoterapéuticas experimentales en el modelo hibrido murino de Reaccion del Injerto contra el Hospedador / Evaluation of experimental immunotherapeutic formulations in the hybrid murine model of Graft-versus-Host Reaction

Introducción: La Enfermedad del Injerto Contra el Hospedador es la complicación más frecuente de los Trasplantes de Células Madre Hematopoyéticas y de todos los trasplantes que contengan células inmunocompetentes alogénicas, el 100 por ciento la padecen y cerca del 30 por ciento mueren por su causa; una proporción alta de casos son esteroide-refractarios, asimismo otras medidas inmunosupresoras modernas fracasan. En los campos de la Inmunoterapia y la Vaccinología también existe una escasez preocupante de inmunomoduladores de origen biológico potentes, efectivos, seguros y de amplio espectro. Existe un modelo híbrido murino de gran utilidad metodológica para estudios experimentales. Objetivo: Evaluar dos formulaciones novedosas de origen biotecnológico, una de ellas inmunopotenciadora y otra inmunosupresora, desarrolladas como cocleatos. Material y Métodos: Mediante Microscopia Electrónica y RT-PCR se caracterizaron las formulaciones como nanopartículas y su capacidad de regular la expresión del ARNm de linfoquinas definitorias de sus perfiles, respectivamente. Empleando el modelo de Enfermedad del Injerto Contra el Hospedador en ratón híbrido F1 (CBAxC57BL), se evaluó su carácter inmunomodulador in vivo . Resultados: Partiendo de los proteoliposomas de Neisseria meningitidis, se obtuvieron dos formulaciones en forma de cocleatos, ambas con diámetros de partícula inferior a 100nm. La Formulación 1mostró un perfil proinflamatorio con potente capacidad de aumentar el IFNγ y el TNFα y potenció el Índice de Bazo hasta 2,05 en el modelo EICH con p=0,0002. La Formulación 2 mostró un perfil supresor-regulatorio con potente capacidad de aumentar la IL-10 y el TGFβ y además de suprimir la producción de TNFα. En el modelo usado, esta formulación, suprimió el Índice de Bazo de manera dosis dependiente y con alta significación estadística. Se corroboró el conocido perfil de seguridad y ausencia de reactogenicidad de ambas formulaciones. Conclusiones: Ambas formulaciones tienen potencial aplicación en los campos de la terapia de Enfermedad del Injerto Contra el Hospedador en otras patologías y en Vaccinología. Los resultados obtenidos en el presente trabajo fundamentan la conveniencia de continuar el desarrollo farmacéutico y completar la preclínica de ambas formulaciones(AU)

Introduction: Graft-versus-host disease is the most frequent complication of Hematopoietic Stem Cell Transplants and all transplants containing allogeneic immunocompetent cells; 100 percent of patients suffer from this complication and about 30 percent die for this particular cause. A high proportion of cases are steroid-refractory; likewise, other modern immunosuppressive measures fail. In the fields of Immunotherapy and Vaccinology, there is also a worrying shortage of powerful, effective, safe and broad spectrum immunomodulators of biological origin. There is a hybrid murine model of great methodological utility for experimental studies. Objective: To evaluate two novel formulations of biotechnological origin: an immunopotentiator formulation and an immunosuppressive one, which were developed as cochleates. Material and Methods: The formulations assayed by Electron Microscopy and RT-PCR were characterized as nanoparticles and for their capacity to regulate lymphokine mRNA expression profile, respectively. The immunomodulatory character was evaluated in vivo using Graft-versus-host disease in (CBAxC57BL) F1 hybrid mice. Results: Starting from the proteoliposomes derived from Neisseria meningitides, two cochleate formulations were obtained, both with particle diameters below 100 nm. Formulation 1 showed a proinflammatory profile with potent capacity to increase IFNγ and TNFα, and potentiated the Spleen Index up to 2.05 in the GVDH model with p = 0.0002. Formulation 2 showed a suppressor/regulatory profile with potent capacity to increase IL-10 and TGFβ and suppress the production of TNFα. In the model used, this formulation suppressed the Spleen Index in a dose-dependent manner with high statistical significance. The known safety profile and absence of reactogenicity of both formulations was corroborated. Conclusions: Both formulations have potential application in the fields of GVHD therapy and other pathologies as well as in Vaccinology. The results obtained in the present work suggest the usefulness to continue with the pharmaceutical development and complete the preclinical studies of both formulations(AU)

Predicting kidney transplant outcomes with partial knowledge of HLA mismatch.

We consider prediction of graft survival when a kidney from a deceased donor is transplanted into a recipient, with a focus on the variation of survival with degree of human leukocyte antigen (HLA) mismatch. Previous studies have used data from the Scientific Registry of Transplant Recipients (SRTR) to predict survival conditional on partial characterization of HLA mismatch. Whereas earlier studies assumed proportional hazards models, we used nonparametric regression methods. These do not make the unrealistic assumption that relative risks are invariant as a function of time since transplant, and hence should be more accurate. To refine the predictions possible with partial knowledge of HLA mismatch, it has been suggested that HaploStats statistics on the frequencies of haplotypes within specified ethnic/national populations be used to impute complete HLA types. We counsel against this, showing that it cannot improve predictions on average and sometimes yields suboptimal transplant decisions. We show that the HaploStats frequency statistics are nevertheless useful when combined appropriately with the SRTR data. Analysis of the ecological inference problem shows that informative bounds on graft survival probabilities conditional on refined HLA typing are achievable by combining SRTR and HaploStats data with immunological knowledge of the relative effects of mismatch at different HLA loci.

IL17A and IL17F genes polymorphisms are associated with histopathological changes in transplanted kidney.

BACKGROUND: Interleukin 17 is a proinflammatory cytokine involved in immune response after allograft transplantation. IL-17 family of proinflammatory cytokines includes IL-17A and IL-17F. Previous studies have demonstrated that the rs2275913 IL17A and the rs11465553 IL17F gene polymorphism are associated with kidney allograft function. Because of the association between these polymorphisms and post-transplant immune response, we assume that these single nucleotide polymorphisms may affect morphological structure of transplanted kidney. The aim of this study was to examine the association of rs2275913 IL17A and rs2397084, rs11465553 and rs763780 IL17F gene polymorphisms with histopathological changes in transplanted kidney biopsies such as: glomerulitis, tubulitis, arteritis, cell infilitration and fibrosis. METHODS: The study enrolled 82 patients after renal graft transplantation in whom a kidney biopsy was performed because of impaired graft function. The rs2397084 T > C (Glu126Gly), rs11465553 G > A (Val155Ile) and rs763780 T > C (His167Arg) polymorphisms within the IL17F gene and the rs2275913 A > G (- 197 A > G) polymorphism within the IL17A gene promoter were genotyped using TaqMan genotyping assays on a 7500 FAST Real-Time PCR System (Applied Biosystems, USA). RESULTS: There was a significant association between the rs2275913 IL17A gene polymorphism and the grade of tubulitis, which was more severe among patients with the A allele, compared to recipients with the GG genotype (GG vs. AG + AA, P = 0.02), and with the grade of arteriolar hyaline thickening and mesangial matrix increase, which were more severe among patients with the G allele compared to recipients with the AA genotype (AA vs. AG + GG, P = 0.01 and P = 0.04, respectively). Tubular atrophy and interstitial fibrosis were more severe among individuals with the C allele at the rs763780 IL17F gene polymorphism (TT vs. TC, P = 0.09 and P = 0.017, respectively). However, it should be taken into account that the statistical significance was achieved without correction for multiple testing, and no significant association would remain significant after such correction. CONCLUSIONS: The results of this study may suggest a possible association between the rs2275913 IL17A and rs2275913 IL17A gene polymorphisms and some histopathological changes in transplanted kidney biopsies.

The genetics of kidney transplantation.

Over the last decade, the search for gene variants with the potential to influence transplant outcomes or predispose individuals to host-recipient-related phenotypes has generated a considerable number of studies with conflicting results. Thousands of genotypes have been associated with complex traits related to transplant medicine, including acute rejection, immunosuppressive drug metabolism and side effects, infections, long-term outcomes, and cardiovascular complications. However, these efforts have given disappointing results, both in terms of gaining understanding of the biological basis of disease and in patient management. The methodological weaknesses that constitute the major limitations of most of these studies have been discussed widely. A new generation of approaches is needed to understand the relationship between gene variants and complex kidney transplantation traits. These approaches should be global, to generate original pathophysiological hypotheses, and should rely on advanced genomic tools, including Genome Wide Association studies and Whole Genome Sequencing technologies. Such enterprises will only be successful with the creation of international consortiums that connect partners in clinical, industrial, and academic transplant medicine.

Dissection of effector pathways in the host-versus-graft response to bone marrow transplantation.

We have dissected the role of the primary cytotoxic pathways Fas-FasL and perforin-granzyme in host-versus-graft reactions after allogeneic bone marrow transplantation. Sublethally irradiated female recipient mice deficient for FasL (B6.gld) or perforin (B6.prf-/-) were transplanted with bone marrow from B6 male donors. Donor engraftment in B6.prf-/- recipients was higher when compared with B6.gld, particularly when assessed by in vivo killing, demonstrating the importance of the perforin pathway over the Fas-FasL pathway. In the absence of both pathways, however, donor bone marrow engraftment was not fully restored identifying a role for an additional pathway(s) in the host-versus-graft response.

Immune responses to gene-modified T cells.

Gene-modified T cells were the first gene therapy tool used in clinical gene transfer trials. After the first applications in immunodeficiency diseases, T cell gene therapy has been extended to HIV infection and cancer. The primary obstacle to successful T cell gene therapy has proven to be the robust immune responses elicited by the gene-modified T cells even in severely immunosuppressed patients. The potent antibody and cytotoxic immune responses have interfered with the expression and persistence of the therapeutic transgene. In this review we will address each of the components of T cell gene therapy -- culture conditions, vector, and transgene -- that have elicited these immune responses and the strategies used to minimize them.

Frequency and targeted detection of HLA-DPB1 T cell epitope disparities relevant in unrelated hematopoietic stem cell transplantation.

The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by the presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB1 donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions.

Chimerism in systemic lupus erythematosus--three hypotheses.

Systemic lupus erythematosus (SLE) is an immune-mediated disease characterized by the presence of autoantibodies and a wide array of clinical symptoms. Despite intensive research, the aetiology of SLE is still unknown and is probably multifactorial. Both genetic and environmental factors have been associated with SLE, but these factors alone are insufficient to explain the onset of SLE. Recently, it has been suggested that chimerism plays a role in the pathogenesis of autoimmune diseases, including SLE. Chimerism indicates the presence of cells from one individual in another individual. In an experimental mouse model, the injection of chimeric cells induces a lupus-like disease. In addition, chimerism is found more often in kidneys of women with SLE than in healthy controls. There are several mechanisms by which chimeric cells could be involved in the pathogenesis of SLE. In this review, three hypotheses on the role of chimerism in SLE are discussed. The first two hypotheses describe the possibilities that chimeric cells induce either a graft-vs-host reaction in the host (comparable with reactions seen after bone marrow transplantation) or a host-vs-graft reaction (comparable with reactions seen after solid organ transplantation). The third hypothesis discusses the possible beneficial role chimeric cells may play in repair mechanisms due to their stem cell-like properties. This review provides insights into the mechanisms by which chimerism may be involved in SLE and proposes several lines of inquiry to further investigate chimerism in SLE.

Mechanisms of the antitumor responses and host-versus-graft reactions induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: a critical role for recipient CD4+ T cells and recipient leukocyte infusion-derived IFN-gamma-producing CD8+ T cells.

Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-gamma production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-gamma-producing cells. RLI-derived CD8 T cells induce the production of IFN-gamma by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-gamma-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.